Project Summary Hematopoietic stem cell (HSC) transplantation (HSCT) represents the only curative therapy for most hematologic disease including leukemia. HSCT requires donor HSCs to engage with the proper supporting niche, survive, proliferate, and differentiate into mature blood lineages. Recent studies suggest that ?stress hematopoiesis?, including that which occurs post-HSCT, is subject to distinct regulation compared steady-state hematopoiesis. A better understanding the molecular mechanisms that regulate transplanted HSCs and their engraftment is necessary to improve HSCT, especially in situations where transplant is limited by small cell numbers (e.g. cord blood) or potentially compromised by ex vivo manipulation (e.g. gene therapy). Towards this, the main goal of this project is to identify and characterize novel key intrinsic regulators of HSCT. My work reveals high expression of multiple GPRASPs (G-Protein Coupled Receptor Associated Sorting Proteins) (e.g. Gprasp1 and Gprasp2) in HSCs, relative to downstream progenitors. I have also found that both GPRASP1 and GPRASP2 act as negative regulators of HSCT. Gprasp-deficient HSCs present enhanced homing, survival and quiescence during transplant. This phenotype appears dependent on Cxcr4, a master regulator of HSCs function, whose sequence contains a putative GASP-binding motif. Indeed, ectopic Cxcr4 enhances HSC homing, survival and quiescence during transplant, phenocopying loss of Gprasp1 or Gprasp2. Further, my preliminary studies show that, upon Gprasp1 or Gprasp2 loss, CXCR4 is stabilized and accumulates on HSCs, sensitizing them to SDF-1. CXCR4 has also been implicated in regulating cell-to-cell contact between B-cell progenitors and stroma cells. Interesting, my recent data suggests that GPRASP loss may also perturb B-cell production by blocking the differentiation of lymphoid progenitors. Finally, although HSCs as a population are highly enriched for Gprasp1 and Gprasp2 expression, my recent analyses of single cell gene expression data and single HSCs by immunofluorescence reveals that the expression of these genes is heterogeneous amongst individual HSCs. This differential expression corresponds with distinct gene expression profiles (e.g. stemness, cell cycle, B-cell differentiation). Although clonal analyses have revealed that even highly purified HSCs display diverse differentiation potential, the molecular regulation of this functional heterogeneity is currently poorly understood. Thus, I hypothesize that multiple GPRASP family members regulate HSC transplantation and B-cell differentiation, and correlate with HSC functional heterogeneity. The mentored phase of this proposal will occur at St. Jude Children?s Research Hospital, under Dr. Shannon McKinney-Freeman. In the independent phase, I will confirm and extend my studies of the molecular link between GPRASPs and CXCR4 in HSCT and B-cell differentiation, as well as exploit novel molecular reporters to refine HSC functional heterogeneity. The institutional resources and academic environment and the planned courses outlined in my proposal will ensure my successful transition to independence.